DNA encoding hydantoinase, DNA encoding N-carbamyl-L-amino acid hydrolase, recombinant DNA, transformed cell, method of producing protein, and method of producing optically active amino acid

Abstract

The present invention provides a method of producing optically active amino acids from 5-substituted hydantoin by isolating a hydantoinase gene and an N-carbamyl-L-amino acid hydrolase gene involved in an ability to convert 5-substituted hydantoin or N-carbamylamino acid into optically active amino acids from a microorganism of the genus Microbacterium having the above ability and by improving gene amplification and transcriptional and translational activities thereby preparing a recombinant wherein the amount of the desired enzymes produced is increased. The hydantoinase gene is, for example, a DNA encoding for a protein having a hydantoinase activity, which has the nucleotide sequence set forth in SEQ ID NO:1 in the Sequence. The N-carbamyl-L-amino acid hydrolase gene is, for example, a DNA encoding for a protein having an N-carbamyl-L-amino acid hydrolase activity, which has the nucleotide sequence set forth in SEQ ID NO:3 in the Sequence.

1. An isolated or purified DNA selected from the following nucleotide sequence (a) or (b):
 (a) the nucleotide sequence comprising SEQ ID NO: 1, and 
 (b) a nucleotide sequence that hybridizes at a salt concentration corresponding to 0.1×SSC, 0.1% SDS at 60° C. with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1, 
 wherein said DNA encodes a polypeptide having hydantoinase activity. 
 
     
     
       2. A recombinant DNA comprising the DNA according to  claim 1  and a vector DNA. 
     
     
       3. The recombinant DNA according to  claim 2 , wherein the vector DNA is a pUC series plasmid, a pBR322 series plasmid, or a plasmid derived from derivatives thereof. 
     
     
       4. A host cell comprising the recombinant DNA according to  claim 3 . 
     
     
       5. The host cell according to  claim 4 , wherein the cell is a  Escherichia coli  bacterium. 
     
     
       6. A method for making a hydantoinase protein, comprising culturing the host cell of  claim 5  for a time and under conditions suitable for expression of a hydantoinase, and collecting the hydantoinase. 
     
     
       7. A method for producing N-carbamylamino acids, comprising contacting a 5-substituted hydantoin with a protein obtained by the process of  claim 6 . 
     
     
       8. A method of producing optically active amino acids, comprising contacting a 5-substituted hydantoin with a protein obtained by the process of  claim 6  and an enzyme optico-selectively hydrolyzing N-carbamylamino acids. 
     
     
       9. A method of producing optically active tyrosine, comprising
 (i) producing a protein having hydantoinase activity by the process of  claim 6  and 
 (ii) producing the optically active tyrosine by contacting DL-5-(4-hydroxybenzyl)hydantoin with: 
 (a) said protein having hydantoinase activity, and 
 (b) an enzyme optico-selectively hydrolyzing N-carbamylamino acids or a material containing the enzyme. 
 
     
     
       10. A method of producing optically active N-carbamylamino acids, comprising:
 (i) producing a protein having hydantoinase activity by the process of  claim 6 ; and 
 (ii) producing the optically active N-carbamylamino acids by contacting a 5-substituted hydantoin with said protein having hydantoinase activity. 
 
     
     
       11. A method of producing optically active N-carbamyltyrosine, comprising:
 (i) producing a protein having hydantoinase activity by the process of  claim 6 ; and 
 (ii) producing the optically active N-carbamyltyrosine by contacting a DL-5-(4-hydroxybenzyl)hydantoin with said protein having hydantoinase activity. 
 
     
     
       12. A method of producing optically active amino acids, comprising chemically or enzymatically converting optically active N-carbamylamino acids produced by the method of  claim 10  into amino acids. 
     
     
       13. A method of producing optically active tyrosine, comprising chemically or enzymatically converting optically active N-carbamyltyrosine produced by the method of  claim 11  into optically active tyrosine. 
     
     
       14. An isolated hydantoinase protein made by the method of  claim 6 . 
     
     
       15. The protein of  claim 14 , wherein said protein has the amino acid sequence of SEQ ID NO: 2. 
     
     
       16. An isolated DNA encoding a protein having a hydantoinase activity, wherein said protein has an amino acid sequence selected from the following amino acid sequence (c) or (d):
 (c) the amino acid sequence of SEQ ID NO:2, and 
 (d) an amino acid sequence wherein in the amino acid sequence of SEQ ID NO:2, one to ten amino acid residues are replaced, deleted, inserted, added or inverted. 
 
     
     
       17. A recombinant DNA comprising the DNA according to  claim 16  and a vector DNA. 
     
     
       18. The recombinant DNA according to  claim 17 , wherein the vector DNA is a pUC series plasmid, a pBR322 series plasmid, or a plasmid derived from derivatives thereof. 
     
     
       19. A host cell comprising the recombinant DNA according to  claim 17 . 
     
     
       20. The host cell according to  claim 19 , wherein the cell is a  Escherichia coli  bacterium. 
     
     
       21. A method for making a hydantoinase protein, comprising culturing the host cell of  claim 19  for a time and under conditions suitable for expression of a hydantoinase, and collecting the hydantoinase. 
     
     
       22. A method for producing N-carbamylamino acids, comprising contacting a 5-substituted hydantoin with a protein obtained by the process of  claim 21 . 
     
     
       23. A method of producing optically active amino acids, comprising contacting a 5-substituted hydantoin with a protein obtained by the process of  claim 21  and an enzyme optico-selectively hydrolyzing N-carbamylamino acids. 
     
     
       24. A method of producing optically active tyrosine, comprising
 (i) producing a protein having hydantoinase activity by the process of  claim 21  and 
 (ii) producing the optically active tyrosine by contacting DL-5-(4-hydroxybenzyl)hydantoin with: 
 (a) said protein having hydantoinase activity, and 
 (b) an enzyme optico-selectively hydrolyzing N-carbamylamino acids or a material containing the enzyme. 
 
     
     
       25. A method of producing optically active N -carbamylamino acids, comprising:
 (i) producing a protein having hydantoinase activity by the process of  claim 21 ; and 
 (ii) producing the optically active N-carbamylamino acids by contacting a 5-substituted hydantoin with said protein having hydantoinase activity. 
 
     
     
       26. A method of producing optically active N -carbamyltyrosine, comprising:
 (i) producing a protein having hydantoinase activity by the process of  claim 21 ; and 
 (ii) producing the optically active N-carbamyltyrosine by contacting a DL-5-(4-hydroxybenzyl)hydantoin with said protein having hydantoinase activity. 
 
     
     
       27. A method of producing optically active amino acids, comprising chemically or enzymatically converting optically active N-carbamylamino acids produced by the method of  claim 25  into amino acids. 
     
     
       28. A method of producing optically active tyrosine, comprising chemically or enzymatically converting optically active N-carbamyltyrosine produced by the method of  claim 26  into optically active tyrosine. 
     
     
       29. A method of producing optically active L -amino acids, comprising;
 (i) producing a protein having hydantoinase activity by culturing a host cell comprising an isolated or purified DNA according to  claim 1  for a time and under conditions suitable for expression of said protein having hydantoinase activity; 
 (ii) producing a protein having N-carbamyl-L-amino acid hydrolase activity by culturing a host cell comprising an isolated or purified DNA selected from the following nucleotide sequence (a) or (b): 
 (a) the nucleotide sequence comprising SEQ ID NO:3, and 
 (b) a nucleotide sequence that hybridizes at a salt concentration corresponding to 0.1×SSC, 0.1% SDS at 60° C. with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:3, 
 for a time and under conditions suitable for expression of said protein having N -carbamyl-L-amino acid hydrolase activity; and 
 (iii) contacting a 5-substituted hydantoin with said protein having hydantoinase activity and said protein having N-carbamyl-L-amino acid hydrolase activity. 
 
     
     
       30. A method of producing optically active L-amino acids, comprising recovering the optically active L-amino acids of  claim 29 . 
     
     
       31. A method of producing optically active L -amino acids, comprising:
 (i) producing a protein having hydantoinase activity by culturing a host cell comprising an isolated or purified DNA according to  claim 1  for a time and under conditions suitable for expression of said protein having hydantoinase activity; 
 (ii) producing a protein having N-carbamyl-L-amino acid hydrolase activity by culturing a host cell comprising an isolated or purified DNA encoding a protein having an N -carbamyl-L-amino acid hydrolase activity, wherein said protein has an amino acid sequence selected from the following amino acid sequence (c) or (d): 
 (c) the amino acid sequence of SEQ ID NO:4, and 
 (d) an amino acid sequence wherein in the amino acid sequence of SEQ ID NO:4, one to ten amino acid residues are replaced, deleted, inserted, added or inverted, 
 for a time and under conditions suitable for expression of said protein having N -carbamyl-L-amino acid hydrolase activity; and 
 (iii) contacting a 5-substituted hydantoin with said protein having hydantoinase activity and said protein having N-carbamyl-L-amino acid hydrolase activity. 
 
     
     
       32. A method of producing optically active L-amino acids, comprising recovering the optically active L-amino acids of  claim 31 . 
     
     
       33. A method of producing optically active L -amino acids, comprising;
 (i) producing a protein having hydantoinase activity by culturing a host cell comprising an isolated or purified DNA according to  claim 16  for a time and under conditions suitable for expression of said protein having hydantoinase activity, 
 (ii) producing a protein having N-carbamyl-L-amino acid hydrolase activity by culturing a host cell comprising an isolated or purified DNA selected from the following nucleotide sequence (a) or (b): 
 (a) the nucleotide sequence comprising SEQ ID NO:3, and 
 (b) a nucleotide sequence that hybridizes at a salt concentration corresponding to 0.1×SSC, 0.1% SDS at 60° C. with DNA consisting of nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:3, 
 for a time and under conditions suitable for expression of said protein having N carbamyl-L-amino acid hydrolase activity; and 
 (iii) contacting a 5-substituted hydantoin with said protein having hydantoinase activity and said protein having N-carbamyl-L-amino acid hydrolase activity. 
 
     
     
       34. A method of producing optically active L-amino acids, comprising recovering the optically active L-amino acids of  claim 33 . 
     
     
       35. A method of producing optically active L -amino acids, comprising:
 (i) producing a protein having hydantoinase activity by culturing a host cell comprising an isolated or purified DNA according to  claim 16  for a time and under conditions suitable for expression of said protein having hydantoinase activity; 
 (ii) producing a protein having N-carbamyl-L-amino acid hydrolase activity by culturing a host cell comprising an isolated or purified DNA encoding a protein having an N -carbamyl-L-amino acid hydrolase activity, wherein said protein has an amino acid sequence selected from the following amino acid sequence (c) or (d): 
 (c) the amino acid sequence of SEQ ID NO:4, and 
 (d) an amino acid sequence wherein in the amino acid sequence of SEQ ID NO:4, one to ten amino acid residues are replaced, deleted, inserted, added or inverted, 
 for a time and under conditions suitable for expression of said protein having N -carbamyl-L-amino acid hydrolase activity; and 
 (iii) contacting a 5-substituted hydantoin with a said protein having hydantoinase activity and said protein having N-carbamyl-L-amino acid hydrolase activity. 
 
     
     
       36. A method of producing optically active L-amino acids, comprising recovering the optically active L-amino acids of  claim 35 . 
     
     
       37. A method of producing optically active L-tyrosine, comprising:
 (i) producing a protein having hydantoinase activity by culturing a host cell comprising an isolated or purified DNA according to  claim 1  for a time and under conditions suitable for expression of said protein having hydantoinase activity; 
 (ii) producing a protein having N-carbamyl-L-amino acid hydrolase activity by culturing a host cell comprising an isolated or purified DNA selected from the following nucleotide sequence (a) or (b): 
 (a) the nucleotide sequence comprising SEQ ID NO:3, and 
 (b) a nucleotide sequence that hybridizes at a salt concentration corresponding to 0.1×SSC, 0.1% SDS at 60° C. with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:3, 
 for a time and under conditions suitable for expression of said protein having N -carbamyl-L-amino acid hydrolase activity; and 
 (iii) contacting a DL-5-(4-hydroxybenzyl)hydantoin with a said protein having hydantoinase activity and said protein having N-carbamyl-L-amino acid hydrolase activity. 
 
     
     
       38. A method of producing optically active L-tyrosine, comprising recovering the optically active L-tyrosine of  claim 37 . 
     
     
       39. A method of producing optically active L-tyrosine, comprising:
 (i) producing a protein having hydantoinase activity by culturing a host cell comprising an isolated or purified DNA according to  claim 1  for a time and under conditions suitable for expression of said protein having hydantoinase activity; 
 (ii) producing a protein having N-carbamyl-L-amino acid hydrolase activity by culturing a host cell comprising an isolated or purified DNA encoding a protein having an N -carbamyl-L-amino acid hydrolase activity, wherein said protein has an amino acid sequence selected from the following amino acid sequence (c) or (d): 
 (c) the amino acid sequence of SEQ ID NO:4, and 
 (d) an amino acid sequence wherein in the amino acid sequence of SEQ ID NO:4, one to ten amino acid residues are replaced, deleted, inserted, added or inverted, 
 for a time and under conditions suitable for expression of said protein having N -carbamyl-L-amino acid hydrolase activity; and 
 (iii) contacting a DL-5-(4-hydroxybenzyl)hydantoin with said protein having hydantoinase activity and said protein having N-carbamyl-L-amino acid hydrolase activity. 
 
     
     
       40. A method of producing optically active L-tyrosine, comprising recovering the optically active L-tyrosine of  claim 39 . 
     
     
       41. A method of producing optically active L-tyrosine, comprising:
 (i) producing a protein having hydantoinase activity by culturing a host cell comprising an isolated or purified DNA according to  claim 16  for a time and under conditions suitable for expression of said protein having hydantoinase activity; 
 (ii) producing a protein having N-carbamyl-L-amino acid hydrolase activity by culturing a host cell comprising an isolated or purified DNA selected from the following nucleotide sequence (a) or (b): 
 (a) the nucleotide sequence comprising SEQ ID NO:3, and 
 (b) a nucleotide sequence that hybridizes at a salt concentration corresponding to 0.1×SSC, 0.1% SDS at 60° C. with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:3, 
 for a time and under conditions suitable for expression of said protein having N -carbamyl-L-amino acid hydrolase activity; and 
 (iii) contacting a DL-5-(4-hydroxybenzyl)hydantoin with a said protein having hydantoinase activity and said protein having N-carbamyl-L-amino acid hydrolase activity. 
 
     
     
       42. A method of producing optically active L-tyrosine, comprising recovering the optically active L-tyrosine of  claim 41 . 
     
     
       43. A method of producing optically active L-tyrosine, comprising:
 (i) producing a protein having hydantoinase activity by culturing a host cell comprising an isolated or purified DNA according to  claim 16  for a time and under conditions suitable for expression of said protein having hydantoinase activity; 
 (ii) producing a protein having N-carbamyl-L-amino acid hydrolase activity by culturing a host cell comprising an isolated or purified DNA encoding a protein having an N -carbamyl-L-amino acid hydrolase activity, wherein said protein has an amino acid sequence selected from the following amino acid sequence (c) or (d): 
 (c) the amino acid sequence of SEQ ID NO:4, and 
 (d) an amino acid sequence wherein in the amino acid sequence of SEQ ID NO:4, one to ten amino acid residues are replaced, deleted, inserted, added or inverted, 
 for a time and under conditions suitable for expression of said protein having N -carbamyl-L-amino acid hydrolase activity; and 
 (iii) contacting a DL-5-(4-hydroxybenzyl)hydantoin with said protein having hydantoinase activity and said protein having N-carbamyl-L-amino acid hydrolase activity. 
 
     
     
       44. A method of producing optically active L-tyrosine acids, comprising recovering the optically active L-tyrosine of  claim 43 . 
     
     
       45. An isolated hydantoinase protein made by the method of  claim 21 . 
     
     
       46. The protein of  claim 45 , wherein said protein has the amino acid sequence of SEQ ID NO: 2.